The present invention relates generally to the modulation of hyaluronan (HA) synthesis and degradation. More particularly, the present invention provides compositions and methods for modulating the expression of genetic material encoding HA synthase (HAS) and other enzymes or receptors primarily involved in hyaluronan metabolism; or modulating the proteins that synthesise or degrade hyaluronan including HAS function or activity. The compositions include or comprise nucleic acid molecules and interactive molecules such as antibodies and small molecule inhibitors and HAS substrate analogs. The present invention relates generally to the treatment and prophylaxis of cellular proliferation and in particular cancer and inflammatory disorders including hyperproliferative conditions. More particularly, the present invention targets hyaluronan anabolism to facilitate control of cellular proliferative conditions such as cancer and inflammation.
Bibliographic details of the publications referred to in this specification are also collected at the end of the description.
Reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the general knowledge in any country.
Inflammatory conditions represent a major causative factor in numerous medically significant disorders. Inflammation can result from a range of stimuli from outside or within the body. However, these stimuli trigger cells and physiological processes within a host environment. Whilst a substantial amount of research has been undertaken to investigate the cellular and cytokine nature of inflammatory processes, less is known about other possible participants in inflammation.
One such class of participants is transmembrane proteins. Transmembrane proteins are involved in a range of signalling activities and many have anzymic activity.
Hyaluronan (HA) metabolism is an intricate balance between the rate of HA synthesis and degradation where depending upon the physiological role being played by the HA, the simultaneous synthesis and degradation is carefully controlled. Hyaluronan is synthesised by a family of distinct yet related transmembrane proteins termed hyaluronan synthase (HAS) isoforms HAS1, 2 and 3, which can be distinguished from one another with respect to temporal and differential expression during mouse embryogenesis and in mature tissues, respectively and also in the molecular weight of the HA produced. The extracellular matrix polysaccharide HA or its acidic form, hyaluronic acid, is a linear, high molecular weight polymer comprised of repeating disaccharide units of (β1-3) D-glucuronate-(β1-4)N-acetyl-D-glucosamine (Weissman & Meyer, J. Am. Chem. Soc. 76: 1753, 1954). Hyaluronan is degraded by a family of enzymes known as hyaluronidases which are currently termed HYAL1, HYAL2, HYAL3 and PH-20, where like the enzymes which produce HA are also distinguished from one another with respect to temporal and differential expression during different physiological processes and disease states.
In work leading up to the present invention, it was observed that HAS and HYAL are differentially expressed under various physiological conditions. In particular, they were up-regulated during disease conditions such as an inflammatory condition or cancer. HAS, and in particular, HAS1, 2 and/or 3 and HYAL1, 2 and/or 3 represent useful drug targets.
The extracellular matrix (ECM) is one of the crucial environmental elements that affect tumor cell behaviour. ECM serves as a scaffold to which tumor cells adhere and migrate as well as acting as a reservoir of growth factors and cytokines that are potentially beneficial to malignant cells (Ruoslahti & Yamaguchi, Cell 64:867-869, 1991). Hyaluronan (HA) is a negatively charged high molecular weight polysaccharide, which is an essential component of the extracellular matrix. For many years HA was considered the “space filler” of the ECM, but with the discovery HA cell surface receptors it has become apparent that it plays a more complex role in cell behaviour (Laurent & Fraser, FASEB J 6:2397-2404, 1992). More recently, HA has been associated with many different cellular hyperproliferative processes including cell division and migration as occurs during development (Toole, J. Internal Medicine 242:35-40, 1981), tissue remodelling (Knudson & Knudson, FASEB J. 7:1233, 1993), inflammation and tumor initiation, progression or invasion (Knudson et al, The Biology of Hyaluronan 143:150-169, 1989).
HAS1, the least active of the three HAS proteins, drives the synthesis of high molecular weight HA (2×106 D), thus HAS1 may play a role in maintaining a low, yet necessary level of HA synthesis in many cell types. HAS2 is widely expressed throughout embryonic development, where it is more active than HAS1 and it also synthesises high molecular weight HA (2×106 D) that is attached to the plasma membrane in the form of a pericellular gel (Fulop, Arch. Iochem. Iophys. 337:261-266, 1997). The HAS2 production of large amounts of high molecular weight HA may have a significant effect on tissue structure and volume, thus playing an important role in developmental processes involving tumor growth. HAS3 is the most active of the 3 HAS proteins, but drives the synthesis of short (<2×105 to 3×105 D) HA chains. HAS3 expression may be activated to produce large amounts of low molecular weight HA, which may interact with cell surface HA receptors, triggering signaling cascades leading to changes in cell behaviour.
HAS2 is important in a highly invasive breast cancer cell line (Udabage et al, Cancer Res 65:6139-6150, 2005) Inhibition of HAS2 using antisense technology resulted in a 97% inhibition of cell invasiveness. Moreover, when antisense HAS2 cancer cells were implanted into nude mice, tumors did not initiate highlighting the potential therapeutic value of inhibiting a functional hyaluronan synthase in the malignant state.
These aforementioned studies highlight the importance of HAS isoforms in relation to cancer where HA synthesis can promote anchorage-independent growth and increase tumor invasiveness, properties that are hallmarks of the malignant phenotype.
The applicant has surprisingly discovered compounds, for example, antibodies, that interact with different sections of HAS amino acid sequence and which specifically target a binding epitope contained within the HAS epitope that is more accessible to an extracellular environment in disease associated cells compared to normal cells.